1. Field of the Invention
The present invention relates to the field of hyperbranched macromolecules. The present invention relates to the field of functionalized substrates on which is bound the macromolecules. The present invention also relates to the field of functionalized size-controlled dendrimers and dendrons that are used to bind to a functionalized substrate at one end of the dendron and to a target-specific ligand on the other end. The present invention also relates to the field of combinatorial chemistry, specific protein detection methods, specific nucleic acid or nucleic acid/peptide hybrid detection methods using a functionalized substrate to which is bound a hyperbranched polymer linked to a probe biomolecule.
2. General Background and State of the Art
Since the first report (Fodor et al., Nature 364, 555-556 (1993); Saiki et al., Proc. Natl. Acad. Sci. USA 86, 6230-6234 (1986)), DNA microarrays have attracted a great deal of attention because they allow high-throughput analysis of the DNA sequence, genetic variations, and gene expression. It is known that this methodology requires improvement in terms of fidelity, reproducibility, and spot homogeneity that are essential for the standardization and application to human gene diagnosis (Hackett et al., Nature Biotechnology 21, 742-743 (2003)). These shortcomings are caused mainly by the variations in the nature of the surface and molecular interlayer structures that are far from ideal. Likewise, the field of high-throughput target detection systems encompasses bioassays utilizing immobilized bioactive molecules and biomolecules.
Here we show that DNA microarrays fabricated on a nanoscale-controlled surface discriminates single mismatched pairs as effectively as DNA does in solution. This approach provides an ideal DNA-microarray in which each probe DNA strand is given ample space enough to interact with an incoming target DNA with minimal steric hindrance. The dramatically increased discrimination efficiency promises the very reliable diagnosis of human genes. Moreover, the approach is general enough to be applied to various bioassays utilizing immobilized bioactive molecules and biomolecules.
Affinity purification is a well-known technique for the separation and identification of ligand-binding proteins (Cuatrecasas et al., Proc. Natl. Acad. Sci. U.S.A. 1968, 61, 636-643). A unique interaction between a ligand covalently attached to an insoluble matrix and the complementary target protein provides the specificity required for the isolation of biomolecules from complex mixtures. However, its widespread use has been hampered by the limited choice and instability of conventional matrices. Significant nonspecific binding of proteins to many solid supports has been a persistent problem in establishing new matrices (Cuatrecasas, P. J. Biol. Chem. 1970, 245, 3059-3065). It is therefore desirable to find new matrices that are comparable to the traditional matrices in terms of the specificity while exhibiting environmental stability and capability of well-defined and facile attachment of ligands.
Aminopropyl-controlled pore glass (or AMPCPG) that is originally used for the solid-phase peptide synthesis appears to have many desirable features. However, the controlled pore glass (or CPG) surface is polar and retains partial negative charge even when coated (Hudson, D. J. Comb. Chem. 1999, 1, 403-457). The feature plays a key role in significant nonspecific binding of proteins. Therefore, application on both affinity chromatography and solid-phase peptide synthesis has been limited. Once the obstacles are eliminated, widespread use of the materials can be expected.
Accessibility of ligands is a key factor in determining binding capacity. The traditional approaches are introducing a spacer molecule and increasing the ligand concentration for better exposition of the ligand on the surface (Rusin, et al., Biosensors & Bioelectronics 1992, 7, 367-373; Suen et al., Ind. Eng. Chem. Res. 2000, 39, 478-487; Penzol et al., Biotechnol and Bioeng. 1998, 60, 518-523; Spinke et al., J. Chem. Phys. 1993, 99, 7012-7019). The approach works to a certain degree, but insufficient space between the ligands and random distribution of capture molecules over the surfaces are the issues yet to be solved (Hearn et al., J. Chromatogr. A. 1990, 512, 23-39; Murza et al., J. Chromatogr. B. 2000, 740, 211-218; Xiao et al., Langmuir 2002, 18, 7728-7739). By far two methods have been employed to improve these shortcomings. One way is to utilize a big molecule such as protein as a placeholder. The protein is conjugated onto the matrix, and the placeholder molecule was cleaved off and washed out. In this way, certain distance between the linkers left on the matrix is secured. Nevertheless, choice of the placeholder molecule and design of the deprotection route have to be elaborately optimized for every different situation (Hahn et al., Anal. Chem. 2003, 75, 543-548). Another way is to employ a cone-shape dendron that gives a highly ordered self-assembled monolayer and utilize an active functional group at the apex of the dendron (Xiao et al., Langmuir 2002, 18, 7728-7739; Whitesell et al., Langmuir 2003, 19, 2357-2365).
Here we present modification of AMPCPG with dendrons, further attachment of GSH at the apex of the dendrons, and characteristics of the surface materials in terms of GST proteins binding. A dendron featuring three or nine carboxylic acid groups at the termini and one amine group at the apex has been introduced into the matrices. Their carboxylic groups were covalently linked with the solid surface. Due to wide use and understating of glutathione S-transferase (or GST) gene fusion system, glutathione was chosen as a ligand to be tethered on the dendron-treated matrix. Ligand binding property of the matrix has been investigated with GST and two fusion proteins (GST-PXP47, GST-Munc-18) (Smith et al., Gene 1988, 67, 31-40; Sebastian et al., Chromatogr. B. 2003, 786, 343-355; Wu et al., Chromatogr. B. 2003, 786, 177-185; De Carlos et al., J. Chromatogr. B. 2003, 786, 7-15).